Exploring the threshold for the start of respiratory syncytial virus infection epidemic season using sentinel surveillance data in Japan.

Introduction: An unusual seasonality of respiratory syncytial virus (RSV) infection in Japan is observed in recent years after 2017, becoming challenging to prepare for: a seasonal shift from autumn-winter to summer-autumn in 2017-2019, no major epidemic in 2020, and an unusually high number of cases reported in 2021. Methods: To early detect the start-timing of epidemic season, we explored the reference threshold for the start-timing of the epidemic period based on the number of cases per sentinel (CPS, a widely used indicator in Japanese surveillance system), using a relative operating characteristic curve analysis (with the epidemic period defined by effective reproduction number). Results: The reference values of Tokyo, Kanagawa, Osaka, and Aichi Prefectures were 0.41, 0.39, 0.42, and 0.24, respectively. Discussion: The reference CPS value could be a valuable indicator for detecting the RSV epidemic and may contribute to the planned introduction of monoclonal antibody against RSV to prevent severe outcomes.

Title: Factors associated with time lag between symptom onset and reporting in the first epidemic wave of COVID-19 in Osaka, Japan.

Objectives: Longer reporting lags after symptom onset reportedly exert a substantial impact on onward transmission, increasing outbreak probability. Our study investigated the risk factors associated with reporting lag. Methods: Using active epidemiological surveillance data for all symptomatic cases reported in Osaka Prefecture during the first wave of the coronavirus disease 2019 (COVID-19) epidemic (February 1-May 13, 2020), multivariable regression analyses were implemented to estimate the effects of exposure variables on reporting lag, by controlling for potential confounders. Results: Cases in their 30s showed a longer reporting lag than cases ≥ 80 years old. Cases who lived in areas with a high COVID-19 incidence demonstrated a longer reporting lag. Cases with a history of visiting a nightlife district also showed longer reporting lag than cases without such a history. Healthcare workers and cases with immunodeficiency both displayed shorter reporting lags than others. Conclusion: Identifying newly infected cases as soon as possible and increased testing capacity for all age groups, and for individuals with a history of visiting high infection-risk areas, represented important measures in shortening reporting lags in the first wave period. The evidence from this study may provide lessons for controlling future emerging diseases.

Seasonal shift in epidemics of respiratory syncytial virus infection in Japan.

In Japan, respiratory syncytial virus (RSV) infection generally has occurred during autumn and winter. However, a possible change in the seasonal trend of RSV infection has been observed recently. The current study was conducted to determine whether the epidemic season of RSV infection in Japan has indeed changed significantly. We used expectation-based Poisson scan statistics to detect periods with high weekly reported RSV cases (epidemic cluster), and the epidemic clusters were detected between September and December in the 2012-2016 seasons while those were detected between July and October in the 2017-2019 seasons. Non-linear and linear ordinary least squares regression models were built to evaluate whether there is a difference in year trend in the epidemic seasonality, and the epidemic season was shifted to earlier in the year in 2017-2019 compared to that in 2012-2016. Although the reason for the shift is unclear, this information may help in clinical practice and public health.

Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

Novel human reovirus isolated from children and its long-term circulation with reassortments.

Mammalian orthoreovirus (MRV), also known as reovirus, was discovered in the 1950s and became the first reported segmented double-stranded RNA virus. MRVs have since been found in a variety of animal species, including humans. However, reports on MRV infections are scarce due to the rarity of their symptomatic occurrence. In Japanese surveillance studies, MRVs have been detected as gastrointestinal pathogens since 1981, with a total of 135 records. In Osaka City, Japan, MRV was first isolated in 1994 from a child with meningitis, and then in 2005 and 2014 from children with gastroenteritis. Here, we conducted the first molecular characterization of human MRV isolates from Japan and identified a novel human reovirus strain belonging to MRV type 2, designated the MRV-2 Osaka strain. This strain, with all three isolates classified, is closely related to MRV-2 isolates from sewage in Taiwan and is relatively close to an MRV-2 isolate from a bat in China. Our data suggest that the MRV-2 Osaka strain, which has circulated amongst humans in Japan for at least two decades, has spread internationally.

GII.17 norovirus infections in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan during two decades.

Norovirus (NoV) is a major cause of viral gastroenteritis, and GII.4 has been the predominant genotype worldwide since the mid-1990s. During the 2014 to 2015 winter, a rare genotype, NoV GII.17, emerged and became prevalent mainly in East Asia. Over the past two decades, NoV molecular surveillance in Osaka City, Japan, has revealed that NoV GII.17 was detected for the first time in February 2001 and that NoV GII.17-associated outbreaks remarkably increased during the 2014 to 2015 season, with higher incidence recorded in January to March 2015. Genetic analysis indicated that 28 GII.17 outbreak strains were closely related to the novel GII.P17-GII.17 variants represented by the Kawasaki308/2015/JP strain, similar to that in other regions. Statistical analysis showed that NoV GII.17 infections were more common in adults than GII.3 and GII.4 infections, suggesting that the affected adults most likely did not have antibodies against NoV GII.17 and the novel GII.17 variant had recently appeared. Regarding transmission, food was one of the most important factors involved in the spread of NoV GII.17 among adults; 61% of GII.17 outbreaks were foodborne, with oysters being the most common vehicle. Interplay between pathogens, hosts, and environmental factors was considered to be important in the 2014 to 2015 NoV GII.17 epidemic.

Dengue Virus in Traveler Returning to Japan from the Democratic Republic of the Congo, 2015.

Dengue fever (DF) is a mosquito-borne disease and a significant global public health problem. Although a few serological surveys in the literature suggest endemic DF in many parts of Africa, DF cases in these countries are generally underreported because of the lack of diagnostic testing and systematic surveillance; thus, little is known about the phylogenetic profile of circulating strains. In April 2015, DF was diagnosed in a Japanese national returning from the Democratic Republic of the Congo (DRC). Dengue virus 1 (DENV-1) RNA was detected in the patient's serum sample using real-time reverse transcription PCR. Phylogenetic analysis of the E gene revealed that the detected DENV-1 strain was classified as genotype V and was closely related, with 100% nucleotide identity, to the strain causing the 2013 DF epidemic in Angola, which is located directly south of the DRC. This is the first report to characterize the circulating DENV strain in the DRC, and the findings indicate that the DENV-1 strain causing the 2013 DF epidemic in Angola was also circulating in the DRC in 2015.

An Epidemic of Hand, Foot, and Mouth Disease Caused by Coxsackievirus A6 in Osaka City, Japan, in 2017.

The second largest epidemic of hand, foot, and mouth disease since 1982 occurred in 2017, which involved 6,173 cases in Osaka City, Japan. The main causative agent was coxsackievirus A6 (CV-A6). Phylogenetic analysis revealed that the detected CV-A6 strains belonged to genetic groups A3 and A4 in clade A.

Broadly reactive real-time reverse transcription-polymerase chain reaction assay for the detection of human sapovirus genotypes.

Sapoviruses are associated with acute gastroenteritis. Human sapoviruses are classified into four distinct genogroups (GI, GII, GIV, and GV) based on their capsid gene sequences. A TaqMan probe-based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay that detects the representative strains of these four genogroups is widely used for screening fecal specimens, shellfish, and environmental water samples. However, since the development of this test, more genetically diverse sapovirus strains have been reported, which are not detectable by the previously established assays. In this study, we report the development of a broader-range sapovirus real-time RT-PCR assay. The assay can detect 2.5 × 107 and 2.5 × 10 1 copies of sapovirus and therefore is as sensitive as the previous test. Analysis using clinical stool specimens or synthetic DNA revealed that the new system detected strains representative of all the 18 human sapovirus genotypes: GI.1-7, GII.1-8, GIV.1, and GV.1, 2. No cross-reactivity was observed against other representative common enteric viruses (norovirus, rotavirus, astrovirus, and adenovirus). This new assay will be useful as an improved, broadly reactive, and specific screening tool for human sapoviruses.

First Complete Genome Sequences of Human Sapovirus Strains Classified as GI.3, GI.4, GI.6, GI.7, and GII.7.

We report here the first complete genome sequences of genotype GI.3, GI.4, GI.6, GI.7, and GII.7 sapovirus strains, detected from fecal samples of acute gastroenteritis patients. Complete or nearly complete genome sequences of all 18 genotypes of human sapoviruses are now available for phylogenetic analysis and primer design.

Molecular surveillance of norovirus, 2005-16: an epidemiological analysis of data collected from the NoroNet network.

BACKGROUND: The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. METHODS: We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. FINDINGS: We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8·2%) sequences belonged to genotype GI, 15 256 (91·7%) to GII, and seven (<0·1%) to GIV.1. During this period, 26 different norovirus capsid genotypes circulated and 22 different recombinant genomes were found. GII.4 drift variants emerged with 2-3-year periodicity up to 2012, but not afterwards. Instead, the GII.4 Sydney capsid seems to persist through recombination, with a novel recombinant of GII.P16-GII.4 Sydney 2012 variant detected in 2014 in Germany (n=1) and the Netherlands (n=1), and again in 2016 in Japan (n=2), China (n=8), and the Netherlands (n=3). The novel GII.P17-GII.17, first reported in Asia in 2014, has circulated widely in Europe in 2015-16 (GII.P17 made up a highly variable proportion of all sequences in each country [median 11·3%, range 4·2-53·9], as did GII.17 [median 6·3%, range 0-44·5]). GII.4 viruses were more common in outbreaks in health-care settings (2239 [37·2%] of 6022 entries) compared with other genotypes (101 [12·5%] of 809 entries for GI and 263 [13·5%] of 1941 entries for GII non-GII.Pe-GII.4 or GII.P4-GII.4). INTERPRETATION: Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions. FUNDING: European Union's Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund.

Ten-Year Surveillance of Measles Virus from 2007-2016 in Osaka City, Japan.

Measles is a highly contagious infection caused by the measles virus (MV). This study performed long-term surveillance in order to survey the prevalence of MV. A total of 417 patients diagnosed with or suspected of having measles were tested for MV between January 2007 and December 2016 in Osaka City, Japan. Reverse transcription-polymerase chain reaction-based testing of clinical specimens showed that 54 patients (12.9%) were MV-positive. An MV epidemic occurred in 2007, in which all detected MV strains were genotype D5, an epidemic strain in Japan at that time. The detected wild-type MV strains in sporadic or outbreak-associated cases since 2011 included genotypes D4, D8, B3, and H1. Three vaccine strains (all genotype A) were also detected. Children ＜10 years of age accounted for 90.0% of the MV-positive patients in 2007. In contrast, adults (≥ 20 years of age) accounted for the majority of MV-positive cases since 2011, as follows: 100%, 50%, 71.4%, 100%, and 87.5% of cases in 2011, 2013, 2014, 2015, and 2016, respectively. The recent high rate of two-dose MV vaccination coverage among children in Japan may have contributed to the reduced risk of MV infection and onset of measles in young persons.

Distinct genetic clades of enterovirus D68 detected in 2010, 2013, and 2015 in Osaka City, Japan.

The first upsurge of enterovirus D68 (EV-D68), a causative agent of acute respiratory infections (ARIs), in Japan was reported in Osaka City in 2010. In this study, which began in 2010, we surveyed EV-D68 in children with ARIs and analyzed sequences of EV-D68 strains detected. Real-time PCR of 19 respiratory viruses or subtypes of viruses, including enterovirus, was performed on 2,215 specimens from ARI patients (<10 years of age) collected between November 2010 and December 2015 in Osaka City, Japan. EV-D68 was identified in 18 enterovirus-positive specimens (n = 4 in 2013, n = 1 in 2014, and n = 13 in 2015) by analysis of viral protein 1 (VP1) or VP4 sequences, followed by a BLAST search for similar sequences. All EV-D68 strains were detected between June and October (summer to autumn), except for one strain detected in 2014. A phylogenetic analysis of available VP1 sequences revealed that the Osaka strains detected in 2010, 2013, and 2015 belonged to distinct clusters (Clades C, A, and B [Subclade B3], respectively). Comparison of the 5' untranslated regions of these viruses showed that Osaka strains in Clades A, B (Subclade B3), and C commonly had deletions at nucleotide positions 681-703 corresponding to the prototype Fermon strain. Clades B and C had deletions from nucleotide positions 713-724. Since the EV-D68 epidemic in 2010, EV-D68 re-emerged in Osaka City, Japan, in 2013 and 2015. Results of this study indicate that distinct clades of EV-D68 contributed to re-emergences of this virus in 2010, 2013, and 2015 in this limited region.

Impact of Coxsackievirus A6 emergence on hand, foot, and mouth disease epidemic in Osaka City, Japan.

Hand, foot, and mouth disease (HFMD) is an acute febrile illness characterized by fever; sore throat; and vesicular eruptions on the hands, feet, and oral mucosa. Until 2010, HFMD was predominantly associated with enterovirus (EV) A71 and coxsackievirus (CV) A16 in Japan. In 2011, CV-A6 emerged as a primary causative agent, causing the largest HFMD epidemic in Japan since 1981. Since then, CV-A6 has caused large HFMD epidemics every 2 years. The phylogenetic analysis of complete Viral Protein 1 (VP1) sequences revealed that most CV-A6 strains detected from 2011 to 2015 in Osaka City were classified into a different clade compared with CV-A6 strains detected from 1999 until 2009. The majority of CV-A6 strains detected in 2011 and most CV-A6 strains detected from 2013 to 2015 were mainly divided into two distinct genetic groups. Each epidemic strain carried unique amino acid substitutions in the presumed DE, EF, and GH loops of the VP1 protein that is exposed on the surface of the virion. There is a possibility that the appearance of substitutions on the surface of the virion and an accumulation of a susceptible population are significant factors in recent HFMD epidemics.

Epidemics of GI.2 sapovirus in gastroenteritis outbreaks during 2012-2013 in Osaka City, Japan.

Sapovirus (SaV) is a causative agent of gastroenteritis in humans in both sporadic cases and outbreaks. During the period from January 2005 to August 2014, SaV was detected in 30 (5.9%) of 510 gastroenteritis outbreaks in Osaka City, Japan using real-time RT-PCR. Seasonal distribution of SaV-associated outbreaks revealed an increase during the 2011-2012 season and the highest frequency of outbreaks during the 2012-2013 season. Genotyping analysis based on the capsid region demonstrated that the most common genotype was GI.2 (36.7%), in which the strains were closely related. The comparison of complete capsid gene sequences with 18 GI.2 strains (7 strains in this study and 11 from GenBank) between 1990 and 2013 showed that GI.2 strains were classified into at least three genetic clusters (1990-2000, 2004-2007, and 2008-2013) with chronologically unique amino acid residues and accumulation of mutations in the predicted P domain, suggesting the one of the causes of emergence and spread of GI.2 strains. This study will also be helpful for understanding the evolutionary mechanism of the SaV genome.

Human parechovirus infections and child myositis cases associated with genotype 3 in Osaka City, Japan, 2014.

Human parechovirus (HPeV) infects humans early in life and typically causes asymptomatic or mild diseases such as gastrointestinal and respiratory illness but sometimes leads to more serious consequences in neonates and young infants. In 2014, we detected HPeV from 38 patients by real-time reverse transcription-PCR in Osaka City, Japan, and 33 HPeV strains were genotyped based on their VP1 sequences. HPeV genotype 3 (HPeV-3) was the most prevalent and accounted for 22 cases (66.7%) followed by nine HPeV-1 (27.3%), one HPeV-2 (3.0%) and one HPeV-4 (3.0%). Phylogenetic analysis revealed that detected HPeV-3 strains were divided into three genetically distinct groups. One was characterized by a novel single amino acid deletion mutation at the N terminus of the 2A protein as well as the VP1 sequence, whereas the others were closely related to HPeV-3 strains detected in Japan in either 2008 or 2011. These HPeV-3 groups were detected from patients with various symptoms including three myositis cases. Recent papers have demonstrated that HPeV-3 was the aetiological agent for epidemic myalgia exclusively among adults from Yamagata Prefecture in Japan. Here, we provide clinical details and episodes of three myositis patients including an adult and two children in Osaka City, Japan. Our results suggest that HPeV-3 is a causative agent of myositis not only in adults but also in children.

Detection and characterization of a human G9P[4] rotavirus strain in Japan.

In a surveillance system in Osaka City, Japan, 48 sporadic rotavirus A (RVA) infections were detected during 2008/2009-2011/2012 seasons. The G/P-genotypes of detected RVAs were G1P[8], G2P[4], G3P[8], G9P[4], and G9P[8]. Although G9P[4] is a rare genotype that had not been reported in Japan, it was the second most prevalent genotype, following G1P[8], and accounted for 35.3% of RVA cases in the 2011/2012 season. Further genotyping revealed that the G9P[4] strain had genotype 2 internal protein genes except for NSP3: G9-P[4]-I2-R2-C2-M2-A2-N2-T1-E2-H2. Among detected RVA strains, G9P[4] and some G9P[8] strains shared high nucleotide identity in VP7 and NSP3 genes. Phylogenetic and BLAST search analyses showed that the G9P[4] strain in Japan shared high nucleotide identity in genotype 2 genes with common G2P[4] strains circulating globally, but was distinct from other G9P[4] strains circulating worldwide. These results suggest that the G9P[4] strain in Japan may have emerged through an independent reassortment between G9P[8] and G2P[4]. Finally, the role of NSP3 protein in the circulating RVA from an amino acid comparison between T1- and T2-type NSP3 is discussed. These findings provide an important insight into less problematic combinations of circulating RVA genes derived from different genotypes.

Relationship between Severity of Aseptic Meningitis and Cerebrospinal Fluid Cytokine Levels.

BACKGROUND: Pediatricians sometimes see patients with severe aseptic meningitis and prolonged fever or severe headache, or both. This condition generally has a good prognosis and is usually treated with supportive therapy. However, there is neither guideline nor consensus for the treatment of patients with severe aseptic meningitis. Here, we investigated the relationship between disease severity and biomarkers. METHODS: The subjects were 32 children aged 0 to 14 years, 23 of whom had aseptic meningitis and 9 of whom were meningitis-free controls. Aseptic meningitis was retrospectively categorized into two subgroups, namely mumps meningitis (MM) and viral meningitis excluding that caused by mumps (EM). We defined a novel aseptic meningitis severity score (AMSS) from the signs and symptoms of aseptic meningitis and thus evaluated disease severity. We analyzed the profiles of cytokines in the patients' cerebrospinal fluid (CSF). RESULTS: The AMSS in MM was significantly higher than that in EM. IL-4, IL-6, IL-8, IL-10, and G-CSF levels in MM and EM CSF were higher than those in control CSF. IFN-γ levels were higher in MM than in controls (p<0.01). IL-10 and IFN-γ levels in MM were higher than those in EM. CONCLUSIONS: MM was more severe than EM. One likely reason is the higher CSF cytokine levels in MM. IFN-γ may be a potentially strong biomarker of MM severity. Our findings would help further understanding

Associations between co-detected respiratory viruses in children with acute respiratory infections.

Viruses are the major etiological agents of acute respiratory infections (ARIs) in young children. Although respiratory virus co-detections are common, analysis of combinations of co-detected viruses has never been conducted in Japan. Nineteen respiratory viruses or subtypes were surveyed using multiplex real-time PCR on 1,044 pediatric (patient age < 6 years) ARI specimens collected in Osaka City, Japan between January 2010 and December 2011. In total, 891 specimens (85.3%) were virus positive (1,414 viruses were detected), and 388 of the virus-positive specimens (43.5%, 388/891) were positive for multiple viruses. The ratio of multiple/total respiratory virus-positive specimens was high in children aged 0-35 months. Statistical analyses revealed that human bocavirus 1 and human adenovirus were synchronously co-detected. On the other hand, co-detections of human parainfluenza virus type 1 (HPIV-1) with HPIV-3, HPIV-3 with human metapneumovirus (hMPV), hMPV with respiratory syncytial virus A (RSV A), hMPV with influenza virus A (H1N1) 2009 (FLUA (H1N1) 2009), RSV A with RSV B, and human rhinovirus and FLUA (H1N1) 2009 were exclusive. These results suggest that young children (<3 years) are highly susceptible to respiratory viruses, and some combinations of viruses are synchronously or exclusively co-detected.